Neutrophils are the most abundant leukocytes in plasma. They are the first cells recruited to injury sites in response to pathogen invasion, and they act in the first line of innate immune defense. Neutrophils have traditionally been considered effector cells for inflammatory response and acute immunity, functioning through intracellular phagocytosis, and using lytic proteases, reactive oxygen species (ROS) and microbicidal proteins for attack of infective agents. Recent studies have shown that neutrophils also possess immunoregulatory capacity by expressing cytokines, chemokines, Fc receptors and complement components, for signaling with other immune cells, such as dendritic cells, B cells and T cells (Mantovani, Cassatella et al. 2011).
Proteases are important effectors of neutrophils. They not only contribute directly to microbicidal activity but also function in the proteolytic processing of chemokines, cytokines and receptors (Pham 2006; Meyer-Hoffert and Wiedow 2010). This modulatory activity is exemplified by the caspase-independent activation of IL-1β and IL-18 by NE, PR3 and CG (Robertson, Young et al. 2006; Guma, Ronacher et al. 2009; Joosten, Netea et al. 2009) or the conversion of anti-inflammatory progranulin to pro-inflammatory granulin by NE and PR3 (Kenssenbrock, 2008). Furthermore, NE has been shown to couple neutrophil-mediated inflammation with the coagulation pathway by cleaving tissue factor pathway inhibitor on Neutrophil Extracellular Traps (NETs).
NETs are released by stimulated neutrophils in a specific form of cell death called NETosis. NETosis is hypothesized to represent a new mechanism of innate immunity mediated by neutrophils in response to pathogen invasion (Brinkmann, Reichard et al. 2004; Remijsen, Kuijpers et al. 2011). It is characterized by the formation of NETs, networks made of decondensed chromatin and anti-microbial proteins and peptides. NETosis, acting at the first line of innate immune defense, represents a new paradigm of cell death that is distinct from apoptosis and necrosis in many aspects. No nuclear fragmentation or membrane blebbing are observed, and activation is independent of caspase activation, yet it does require NADPH oxidase and MAPK kinase pathways. NETosis also involves activities of NE, myeloperoxidase, and peptidylarginine deiminase 4, an enzyme responsible for histone citrullination and chromatin decondensation (Wang, Li et al. 2009; Papayannopoulos, Metzler et al. 2010). The primary function of NETs is hypothesized to trap and kill pathogens. In addition, it also provides a matrix for high local concentrations of effectors and mediators for the ensuing innate and adaptive immune responses. Previous proteomic studies of NET components identified three major proteases, namely neutrophil NE, CG and PR3 (Urban, Ermert et al. 2009).
In order to characterize NET-associated proteolytic activities in an unbiased manner, proteins trapped in NETs were released and assayed with the Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS) method (O'Donoghue et al., 2012). This method utilizes a library of 124 highly diversified peptides in a multiplex assay with tandem liquid chromatography-mass spectrometry for detection of cleavage sites. Using the MSP-MS assay, the contribution of each enzyme in the complex NETosis sample was deconvoluted by comparison with substrate specificity profiles from purified human neutrophil proteases. In addition, the non-prime side substrate specificity of NE, CG, PR3 and NSP4 was investigated using a tetrapeptide fluorescent substrate library. Reported here is the first complete study, to the inventors' knowledge, that compares the extended substrate specificity of NE, PR3, CG and NSP4 in parallel under identical conditions. Through the analysis of NET-associated protease mixtures from three independent healthy donors, the major activity could be attributed to NE. Immunodepletion of NE activity revealed contributing activity from PR3 and to a lesser extent CG, as well as a trace of NSP4 activity. Identifying the substrate specificity and the contribution of each NET-associated protease to overall NET-associated activity could lead to the development of improved therapeutic intervention for pathological NETosis in acute and chronic immune diseases.